Hindawi Publishing Corporation International Journal of Genomics Volume 2015, Article ID 567809, 12 pages http://dx.doi.org/10.1155/2015/567809
Research Article Comparative Genomics Revealed Genetic Diversity and Species/Strain-Level Differences in Carbohydrate Metabolism of Three Probiotic Bifidobacterial Species Toshitaka Odamaki, Ayako Horigome, Hirosuke Sugahara, Nanami Hashikura, Junichi Minami, Jin-zhong Xiao, and Fumiaki Abe Food Science and Technology Institute, Morinaga Milk Industry Co., Ltd., No. 1-83, 5-Chome Higashihara, Zama, Kanagawa, Japan Correspondence should be addressed to Toshitaka Odamaki;
[email protected] Received 30 December 2014; Revised 4 May 2015; Accepted 26 May 2015 Academic Editor: John Parkinson Copyright © 2015 Toshitaka Odamaki et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Strains of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis are widely used as probiotics in the food industry. Although numerous studies have revealed the properties and functionality of these strains, it is uncertain whether these characteristics are species common or strain specific. To address this issue, we performed a comparative genomic analysis of 49 strains belonging to these three bifidobacterial species to describe their genetic diversity and to evaluate species-level differences. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve. Further analysis focused on carbohydrate metabolism revealed the existence of certain strain-dependent genes, such as those encoding enzymes for host glycan utilisation or certain membrane transporters, and many genes commonly distributed at the species level, as was previously reported in studies with limited strains. As B. longum and B. breve are human-residential bifidobacteria (HRB), whereas B. animalis is a non-HRB species, several of the differences in these species’ gene distributions might be the result of their adaptations to the nutrient environment. This information may aid both in selecting probiotic candidates and in understanding their potential function as probiotics.
1. Background The genus Bifidobacterium has been classified into 48 taxa, including 39 species and nine subspecies [1], three of which (Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis) are commonly applied as bifidobacterial probiotics in the food industry. Many studies have been performed on the functionality of probiotic bifidobacteria in the host, and the properties and functionality of probiotics have generally been thought to be strain dependent [2, 3]. However, previous reports have indicated that certain characteristics, such as the ability to generate vitamins [4], organic acids [5], and antimicrobial components [6, 7] as well as tolerance to stress [8, 9], are species dependent, although there is a degree of variation among strains. In several cases, it
is unclear whether certain characteristics are species common or strain specific. Recent work has provided genomic information for all bifidobacterial species/subspecies [1, 10]. However, because the number of available genomic sequences from each species was limited in these studies, it is difficult to distinguish whether a number of unique genes are species or strain specific. To further elucidate bifidobacterial genetic diversity and to evaluate species-level differences, we performed comparative genomic analyses of 49 strains belonging to three bifidobacterial species, or B. longum, B. breve, and B. animalis, which are commonly applied as probiotics in the food industry. The use of a large set of genome sequences based on a relatively large number of strains of each species allowed the
2 identification of pan-genome structures and the elucidation of differences in core genome structures among these species. In the present report, given that bifidobacteria have been reported to utilise many unique metabolic pathways for sugar fermentation [11–19], additional analysis was focused on the carbohydrate transport/metabolism of each species/strain.
2. Materials and Methods 2.1. Bacterial Strains and Cultivation Conditions. The bacterial strains used in this study and general information about these strains are listed in Table 1. The bifidobacterial strains were obtained from stock cultures maintained in the Morinaga Culture Collection (MCC; Morinaga Milk Industry Co., Ltd., Zama, Japan) and the American Type Culture Collection (ATCC, VA). Each strain was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% Lcysteine⋅HCl (Kanto Chemical, Tokyo, Japan) at 37∘ C for 16 h under anaerobic conditions before DNA extraction. The microorganisms were collected by centrifugation, washed once with sterile saline, resuspended in an equivalent volume of sterile saline, and used as seed cultures for fermentation studies. 2.2. Assimilation of Human Milk Oligosaccharides. Modified MRS was prepared by removing glucose from the original components and was supplemented with LNT (Dextra Laboratories Ltd., Reading, UK) at a final concentration of 2%. An aliquot of seed culture from each bifidobacterial strain was then inoculated into 200 𝜇L of the modified MRS, with a final concentration of 1%. Growth of each bifidobacterial strain was measured by absorbance at OD600 after cultivation under anaerobic conditions at 37∘ C for 24 h and 48 h. Experiments were performed in triplicate. 2.3. Genome Sequencing and Bioinformatics Analyses. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The preparation of genomic libraries was performed with 1 ng of genomic DNA using the Nextera XT DNA Sample Preparation Kit (Illumina Inc., CA) according to the manufacturer’s instructions. After PCR amplification and cleanup, the fragment size distribution of the tagmented DNA was analysed using an Agilent 2100 Bioanalyzer and the High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, CA). The libraries were sequenced using a MiSeq Personal Sequencing System and the MiSeq Reagent Kit v2 (500 cycles) (Illumina Inc.). Quality trimming and de novo assembly of the raw paired-end reads were performed using the CLC Genomics Workbench (v 6.0) software package (CLC bio, Aarhus, Denmark) with default settings, except for contig length (minimum contig length = 2,000 bp). Seventeen genomes that were sequenced de novo in this study were automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 2.0 and were manually checked as part of the process of genome submission to GenBank. Our sequencing efforts resulted in multiple contigs (Table 1). The datasets for the genome annotations for the
International Journal of Genomics other 32 strains published previously were retrieved from the FTP server of NCBI [20]. 2.4. Pan-Genome Analysis. For all of the 49 bifidobacterial strains included in this study, a pan-genome calculation was performed using the PGAP [21]. The ORF content of each genome was organised into functional gene clusters using the gene family (GF) method. Under the GF method, the total protein sequences of each strain were mixed together, with each gene being marked as a strain identifier. BLASTALL searches were performed among the mixed protein sequences, and the filtered BLAST results were clustered using the Markov Cluster algorithm [22], which has been widely used in other studies on prokaryotic genomes and in programs designed to search for orthologues among multiple strains. For each gene pair in a given cluster, the global match region was no less than 50% of the longer gene protein sequence, and the identity was also no less than 50%. The minimum score value and 𝐸-value applied in BLAST were 50 and 1e-8. Pan-genome profile analysis, genetic variation analysis of functional genes, and function enrichment analysis of gene clusters were then performed. KEGG Orthology (KO) numbers were assigned by the KEGG Automatic Annotation Server (KAAS) using the bidirectional best-hit method [23]. 2.5. Phylogenetic Trees. Pan-genome-based phylogenetic trees were generated according to a gene distance matrix, which was calculated based on genes that were absent or present in each strain [21], using UPGMA algorithms in PHYLIP. The sequences of 16S rRNA genes were aligned using the ClustalW alignment tool [24] with default parameters, and phylogenetic trees were constructed using UPGMA algorithms in MEGA6.06 [25]. Bootstrap values were calculated using 500 bootstrap replicates. The supertree was built using FigTree v1.4.0. 2.6. Nucleotide Sequence Accession Numbers. The de novo sequence and annotation data reported herein have been deposited in GenBank under the accession numbers AVQA00000000-AVQE00000000 and AWFK00000000AWFV00000000.
3. Results and Discussion 3.1. Phylogenetic Trees for Each Bifidobacterial Species. Based on the pan-genome profiles and 16S rRNA gene sequences, phylogenetic trees were constructed for each species, using Gardnerella vaginalis ATCC 14018 as an outgroup species. Each phylogenetic tree was divided into two clusters. However, the classification of subspecies of certain strains based on the genotypes of the strains was contradictory to that previously defined based on phenotypes (Table 1, Supplementary files 1–3 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/567809). B. animalis subsp. animalis ATCC 27536 and ATCC 27674 fell into the clade with the type strain of B. animalis subsp. lactis, whereas B. longum subsp. longum JDM 301 and B. longum subsp. infantis 157F fell into the clades with the type strains of B. longum
longum
longum
longum
longum
longum
longum longum longum longum longum longum longum breve breve breve breve breve breve breve breve breve breve breve breve breve breve animalis animalis animalis animalis animalis
B. longum ssp. infantis B. longum ssp. longum B. longum ssp. longum
B. longum ssp. infantis
B. longum ssp. longum
B. longum ssp. longum
B. longum
B. longum
B. longum ssp. longum B. longum ssp. longum B. longum ssp. longum B. longum ssp. longum B. longum ssp. longum B. longum ssp. longum B. longum ssp. longum B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. breve B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis B. animalis ssp. animalis
longum longum longum longum longum longum longum breve breve breve breve breve breve breve breve breve breve breve breve breve breve animalis animalis animalis animalis animalis
longum
longum
longum
longum
longum
Genotype based on 16SrRNA Whole genes infantis infantis infantis infantis longum longum
Species
F8 ATCC 55813 CCUG 52486 1-6B 2-2B 35B 44B ACS-071-V-Sch8b UCC2003 DSM 20213 CECT 7623 MCC0121 MCC0305 MCC0476 MCC1094 MCC1114 MCC1128 MCC1340 MCC1454 MCC1604 MCC1605 ATCC 25527 ATCC 27672 MCC0483 MCC0499 MCC1489
DJO10A
NCC2705
KACC 91563
JCM 1217
157F
ATCC 15697 JDM301 BBMN68
Strain
Complete Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Complete Complete Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Unfinished Complete Unfinished Unfinished Unfinished Unfinished
Complete
Complete
Complete
Complete
Complete
Complete Complete Complete
Status
1
1 1
1
1
1
1
1
1
1 1 1
2
1
2
2
9 27 11 14
117 34 27 22 15 23 33 25 23 14 15 37
140 55 171 141 131 62
2,385 2,373 2,453 2,686 2,625 2,514 2,559 2,327 2,423 2,298 2,314 2,436 2,287 2,234 2,327 2,487 2,480 2,373 2,457 2,206 2,324 1,933 1,990 2,176 2,134 1,910
2,390
2,260
2,396
2,385
2,409
2,833 2,478 2,266
Source
1,681 2,109 2,240 2,425 2,412 2,260 2,262 1,826 1,852 2,263 1,725 2,079 1,927 1,851 1,944 2,136 2,143 1,986 2,111 1,830 1,976 1,538 1,532 1,750 1,721 1,474
2,000
1,728
1,985
1,924
1,999
Human feces Human infant faeces Human elderly faeces Human children feces Human children feces Human infant faeces Human infant faeces Human vagina Human infant faeces Human infant faeces Human milk Human infant faeces Human adult faeces Human adult faeces Human infant faeces Human infant faeces Human infant faeces Human infant faeces Human infant faeces Human elderly faeces Human elderly faeces Rat feces Rat feces Rat feces Rat feces Guinea pig feces
Human adult faeces
Human infant faeces
Human infant faeces
Human infant faeces
Human infant faeces
2,416 Human infant faeces 1,958 Human vagina 1,804 Human elderly faeces
Chromosome Plasmid Contig Length (kbp) CDSs
Table 1: General genome features of Bifidobacterium species included in this study. GenBank accession number CP001095 CP002010 CP002286 AP010890, AP010891, and AP010892 AP010888 CP002794, CP002795, and CP002796 AE014295 and AF540971 CP000605, AF538868, and AF538869 FP929034 ACHI00000000 ABQQ00000000 AJTF00000000 AJTJ00000000 AJTI00000000 AJTM00000000 CP002743 CP000303 ACCG00000000 AFVV00000000 AVQA00000000 AWFR00000000 AVQB00000000 AWFS00000000 AVQC00000000 AVQD00000000 AWFT00000000 AWFU00000000 AVQE00000000 AWFV00000000 CP002567 AWFQ00000000 AWFK00000000 AWFN00000000 AWFO00000000
International Journal of Genomics 3
Genotype based on Species Strain 16SrRNA Whole genes B. animalis ssp. lactis lactis lactis AD011 B. animalis ssp. animalis lactis lactis ATCC 27536 B. animalis ssp. animalis lactis lactis ATCC 27673 B. animalis ssp. animalis lactis lactis ATCC 27674 B. animalis ssp. lactis lactis lactis B420 B. animalis ssp. lactis lactis lactis BB-12 B. animalis ssp. lactis lactis lactis Bi-07 B. animalis ssp. lactis lactis lactis Bl-04 B. animalis ssp. lactis lactis lactis Bl12 B. animalis ssp. lactis lactis lactis BLC1 B. animalis ssp. lactis lactis lactis BS01 B. animalis ssp. lactis lactis lactis CNCM I-2494 B. animalis ssp. lactis lactis lactis DSM 10140 B. animalis ssp. lactis lactis lactis HN019 B. animalis ssp. lactis lactis lactis V9 Complete Unfinished Unfinished Unfinished Complete Complete Complete Complete Complete Complete Unfinished Complete Complete Unfinished Complete
Status
1
1 1
1 1 1 1 1 1
1
1
28
7
18 21 18
1,934 1,912 1,937 1,912 1,939 1,942 1,939 1,939 1,938 1,944 1,932 1,943 1,938 1,916 1,944
Source
1,527 Human infant faeces 1,561 Chicken feces 1,576 Sewage 1,561 Rabbit feces 1,561 No information 1,642 Yoghurt 1,597 No information 1,567 Human infant faeces 1,518 Colonoscopic sample 1,518 No information 1,572 No information 1,660 No information 1,565 Yoghurt 1,578 No information 1,572 Human infant faeces
Chromosome Plasmid Contig Length (kbp) CDSs
Table 1: Continued. GenBank accession number CP001213 AWFL00000000 AWFP00000000 AWFM00000000 CP003497 CP001853 CP003498 CP001515 CP004053 CP003039 AHGW00000000 CP002915 CP001606 ABOT00000000 CP001892
4 International Journal of Genomics
International Journal of Genomics
5
B. longum 23
166
9 584
254
38 B. breve
4 B. animalis
Figure 1: Venn diagram of the homologous clusters shared among the core genes. The surfaces are approximately proportional to the number of genes.
subsp. infantis and B. longum subsp. longum, respectively. In this study, we adopted subspecies of these strains based on the phylogenetic trees for further analyses. 3.2. Core and Pan-Genome Structures. After clustering the functional genes for each species, respective totals of 5,471, 4,053, and 2,833 clusters and 966, 1,221, and 1,092 core clusters were obtained for B. longum, B. breve, and B. animalis (Supplementary file 4). These numbers are similar to those reported previously [11, 26, 27]. However, we identified a wider variety of genomes in B. longum compared with a previous report [11]. This result may suggest that B. longum has an open pan-genome; that is, B. longum may exhibit a robust ability to import new genes, allowing it to adapt to each ecological niche over its long history of evolution. In the current study, to reveal differences among species, a total of 90,442 genes from the 49 strains were jointly clustered. These genes were divided into 8,818 homologous clusters, and there were 584 common clusters among all of the bifidobacterial strains (Figure 1). Additional analysis revealed that 404 of these clusters were commonly clustered with G. vaginalis ATCC 14018 as an outgroup species. Therefore, the number of specific gene clusters among the three bifidobacterial species was estimated to be less than 180. There were 166 common clusters between strains of B. breve and B. longum, whereas there were nine common clusters between strains of B. animalis and B. longum and four common clusters between strains of B. animalis and B. breve (Figure 1). 3.3. Metabolism of Host Glycans. Bifidobacteria have been reported to employ many unique metabolic pathways for sugar fermentation to enable them to utilise diverse carbohydrates in the intestine that are not utilised by their hosts [11–19]. Figure 2 shows the distributions of genes involved in the metabolism of host glycans, such as human milk oligosaccharides (HMOs), mucin, and N-glycans. 3.3.1. HMOs. A 43 kb cluster in the genome of B. longum subsp. infantis ATCC 15697 has been reported to be one of the
most characteristic gene clusters for the utilisation of HMOs [28]. Seven homologous genes encoding extracellular solutebinding proteins predicted to bind oligosaccharides (SBP family 1) were found in only two strains of B. longum subsp. infantis, whereas homologues of the major facilitator superfamily (Blon 2331, 2332) were found in all strains except for B. animalis subsp. lactis AD011. Two of four gene homologues encoding glycoside hydrolases (alpha-galactosidase and betaN-acetylhexosaminidase) were found in strains of B. breve as well as B. longum subsp. infantis. Fucosidase (Blon 2336) and sialidase (Blon 2348) homologues were found only in B. longum subsp. infantis. However, certain strains of B. breve possessed other gene clusters related to fucose and sialic acid incorporation (Figure 2). Regarding the core structure of HMOs, three of the four predominant HMOs (lacto-N-fucopentaose I (LNFP I), lacto-N-difucohexaose I (LNDFH I), and lacto-N-tetraose (LNT)) exhibit the LNT structure. Therefore, the enzyme required for the utilisation of LNT is crucial in the utilisation of HMOs and the colonisation of the infant intestine. Certain strains belonging to B. bifidum, B. longum subsp. longum, B. longum subsp. infantis, and B. breve have been reported to act as LNT consumers [29, 30]. The last two species incorporate intact LNT via an unidentified transporter and then hydrolyse it intracellularly using LNT 𝛽1,3-galactosidase (Blon 2016) [31]. In our study, using type strains, we confirmed the ability of B. longum subsp. longum, B. longum subsp. infantis, and B. breve, but not B. animalis subsp. animalis and B. animalis subsp. lactis, in the utilisation of LNT (Supplementary file 5). Unexpectedly, Blon 2016 homologues were also present in B. animalis strains. Based on phylogenetic analysis, Yoshida et al. [31] discovered that there is a close homologue of this gene (amino acid identity > 95%) that falls into a clade of strains of infant gut-related species (i.e., B. breve, B. longum subsp. infantis, and B. longum subsp. longum), whereas the gene in B. animalis was observed to be distant from this clade. These results imply the existence of different substrate specificity for LNT 𝛽-1,3galactosidase, although further investigations are necessary to examine this issue. B. bifidum and certain strains of B. longum subsp. longum produce a secretory enzyme, LNBase, that hydrolyses LNT into lacto-N-biose (LNB) and lactose [32, 33]. In the present study, homologous genes encoding LNBase (BLLJ 1506) and the chaperone for this enzyme (BLLJ 1505) were found in two strains of B. longum subsp. longum. The LNBase of B. longum subsp. longum has been reported to be able to hydrolyse the GlcNAc𝛽1-3Gal linkage in LNT, LNFP I, and sialyllacto-N-tetraose. These results imply the existence of different mechanisms for the utilisation of LNT decorated by fucose and sialic acid. B. bifidum might digest LNT after releasing its own fucosidase and sialidase, whereas B. longum subsp. longum lacks these enzymes but can directly degrade decorated LNT. Subsequently, the liberated LNB is imported into the cells via the galacto-N-biose (GNB)/LNB transporter for further degradation [34]. The disaccharide is then phosphorolysed by a GNB/LNB phosphorylase to produce Gal-1-P and GlcNAc
6
International Journal of Genomics BRE
ANI
LAC
ATCC25527 ATCC_27672 MCC_0483 MCC_0499 MCC_1489 AD011 ATCC_27536 ATCC_27673 ATCC_27674 B420 BB_12 Bi_07 Bl12 Bl_04 BLC1 BS01 CNCMI_2494 DSM10140 HN019 V9
Description 43 kbp HMO cluster Galactoside symporter Galactoside symporter Integrase Beta-galactosidase Alpha-galactosidase Alpha-L-fucosidase RbsD or FucU transport Dihydrodipicolinate synthase Short chain dehydrogenase Mandelate racemase Hypothetical protein Sugar ABC transporter permease Sugar ABC transporter permease Family 1 extracellular solute-binding protein Sugar ABC transporter permease Sugar ABC transporter permease Family 1 extracellular solute-binding protein Exo-alpha-sialidase N-Acetylneuraminate lyase Family 1 extracellular solute-binding protein Family 1 extracellular solute-binding protein Family 1 extracellular solute-binding protein Hypothetical protein Family 1 extracellular solute-binding protein Beta-N-acetylhexosaminidase Haloacid dehalogenase domain-containing protein hydrolase Family 1 extracellular solute-binding protein Beta-lactamase Binding-protein-dependent transport system Inner membrane protein Binding-protein-dependent transport system Inner membrane protein Sugars ABC transporter ATP-binding protein Sialidase cluster LacI-type transcriptional regulator Transcriptional regulator, GntR family UDP-N-acetylglucosamine diphosphorylase ROK family transcriptional regulator N-Acetylmannosamine-6-phosphate 2-epimerase, NanE Sialidase A Oligopeptide-binding protein oppA Oligopeptide transport system permease protein fusion between oligopeptide transport permease oppC and ATP-binding protein oppD Oligopeptide transport ATP-binding protein N-Acetylneuraminate lyase Fucosidase cluster Two-component response regulator Two-component sensor protein MFS transporter MFS transporter permease Alpha-L-fucosidase LacI-type transcriptional regulator MFS transporter permease Alpha-L-fucosidase Permease of ABC transporter for sugars Sugar ABC transporter permease ABC transporter substrate-binding protein Mandelate racemase/muconate lactonizing domain-containing protein MFS transporter permease 𝛼-fucosidase LNT related gene LNT 𝛽-1,3-galactosidase Chaperon for lacto-N-biosidase Lacto-N-biosidase GNB/LNB cluster Phosphotransferase family Lacto-N-biose phorylase Permease protein of ABC transporter system for sugars Solute-binding protein of ABC transporter system for sugars Galactose-1-phosphate uridylyltransferase Permease protein of ABC transporter system for sugars UDP-glucose 4-epimerase GluNAc/GalNAc related gene Glucosamine-6-phosphate isomerase N-Acetylglucosamine-6-phosphate deacetylase Beta-hexosaminidase A Glycosyl hydrolase PTS system, glucose-specific IIABC component PTS system, N-acetylglucosamine-specific IIBC component Phosphoenolpyruvate-protein phosphotransferase Phosphocarrier protein HPr Endo-beta-N-acetylglucosaminidase Endo-alpha-N-acetylgalactosaminidase 𝛼-N-acetylgalactosaminidase Endo-beta-N-acetylglucosaminidase Endo-beta-N-acetylglucosaminidase 𝛼-mannose related gene Alpha-mannosidase Alpha-mannosidase Solute-binding protein for manno-
INF
1_6B 2_2B 35B 44B 157F ATCC55813 BBMN68 CCUG52486 DJO10A F8 JCM1217 KACC91563 NCC2705 ATCC15697 JDM301 ACS_071_V_Sch8 CECT7263 DSM20213 MCC_0121 MCC_0305 MCC_0476 MCC_1094 MCC_1114 MCC_1128 MCC_1340 MCC_1454 MCC_1604 MCC_1605 UCC2003
LON
Reference Blon_2331 [28] Blon_2332 [28] Blon_2333 [28] Blon_2334 [28] Blon_2335 [28] Blon_2336 [28] Blon_2337 [28] Blon_2338 [28] Blon_2339 [28] Blon_2340 [28] Blon_2341 [28] Blon_2342 [28] Blon_2343 [28] Blon_2344 [24, 28] Blon_2345 [28] Blon_2346 [28] Blon_2347 [24, 28] Blon_2348 [28] Blon_2349 [28] Blon_2350 [24, 28] Blon_2351 [24, 28] Blon_2352 [24, 28] Blon_2353 [28] Blon_2354 [24, 28] Blon_2355 [28]
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2
0
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0
1
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3
2
0
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0
1
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6
1
0
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0
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3
2
0
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6
1
0
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1
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1
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1
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1
1
1
1
1
1
1
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1
1
1
1
1
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0
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0
1
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0
1
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2359 [28]
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2360 [28]
2
2
2
2
2
2
2
2
2
2
2
2
2
4
2
3
2
3
2
2
2
3
2
2
2
2
2
2
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Blon_2361 [28]
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
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0
1
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
2
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
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0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
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0
0
0
0
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0
0
0
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0
0
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0
1
0
1
0
1
1
1
1
0
1
1
1
1
1
1
1
0
0
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0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
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0
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0
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0
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
1
0
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0
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1
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1
1
1
0
0
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0
0
0
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0
1
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2
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1
1
1
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1
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1
1
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1
1
1
1
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1
1
0
1
1
1
0
1
1
0
1
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0
1
1
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0
1
1
1
0
1
1
1
0
1
1
0
1
0
0
1
1
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1
0
1
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1
1
1
0
1
1
0
1
0
0
1
1
0
0
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3
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1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
1
0
0
0
0
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1
1
1
1
1
0
2
1
1
1
2
0
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
2
2
3
1
2
3
2
1
1
3
2
2
3
3
2
2
2
0
4
2
2
1
3
0
1
1
3
0
2
3
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
0
0
0
0
0
0
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1
1
0
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0
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0
0
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0
1
0
0
0
0
0
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0
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0
0
0
0
0
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0
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0
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0
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0
0
0
0
0
0
0
1
0
0
0
0
0
0
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0
0
0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2356 [28] Blon_2357 [28] Blon_2358 [28]
Blon_0641 [28] Blon_0642 [28] Blon_0643 [28] Blon_0644 [28] Blon_0645 [28] Blon_0646 [28] Blon_0647 [28] Blon_0648 [28] Blon_0649 [28] Blon_0650 [28] Blon_0651 [28] Blon_0243 [28] Blon_0244 [28] Blon_0245 [28] Blon_0247 [28] Blon_0248 [28] Blon_0423 [28] Blon_0425 [28] Blon_0426 [28] Blon_0341 [25] Blon_0342 [25] Blon_0343 [25] Blon_0344 [25] Blon_0345 [25] Blon_0346 [25, 26] Blon_2016 [31] BLLJ_1505 [33] BLLJ_1506 [33]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
0
0
0
1
0
0
0
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0
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0
0
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0
0
0
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0
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0
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0
0
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0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2173 [34]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2174 [34]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2175 [34]
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2176 [34]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2177 [34]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
3
2
2
3
2
2
3
3
3
3
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
2
1
1
1
2
1
1
1
2
2
2
2
2
1
1
2
1
1
2
2
2
2
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
2
2
2
2
1
2
2
2
2
2
2
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
1
1
0
1
1
1
2
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_2171 [34] Blon_2172 [34]
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
1
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
0
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
1
1
0
0
1
0
0
0
0
1
0
0
0
0
0
1
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
2
2
2
2
0
3
2
0
0
3
2
2
2
2
2
3
2
2
3
2
2
2
2
2
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
2
2
2
2
0
3
2
0
0
3
2
2
2
2
2
3
2
2
3
2
2
2
2
2
2
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
0
1
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Blon_0881 [24, 25] Blon_0882 [24, 25] Blon_0732 [27] Blon_2468 [42] Blon_2470 [42] Blon_2471 [42] Blon_0178 [42] Blon_0177 [42] BLD_0197 [42] BLD_1258 [39] NagBb [38] — — Blon_0868 [42] Blon_0869 [42] Blon_2380 [42]
Figure 2: Distribution of genes involved in the metabolism of host glycans, such as human milk oligosaccharides, mucin, and N-glycans. The abbreviations used for species/subspecies are as follows: INF, B. longum subsp. infantis; LON, B. longum subsp. longum; BRE, B. breve; ANI, B. animalis subsp. animalis; LAC, B. animalis subsp. lactis.
International Journal of Genomics
7
GH5 GH13 GH27 GH30 GH31 GH36 GH43 GH51 GH53 GH121 GH127 CBM6 CBM10 CBM13 CBM22 CBM23 CBM61
V9
HN019
BS01
DSM10140
BLC1
CNCMI_2494
Bl12
Bl_04
Bi_07
B420
BB_12
ATCC_27674
1
2
2
2
2
2
2
1
2
2
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
10
11
11
10
11
11
11
11
11
9
11
11
8
9
11
11
9
10
10
10
11
10
11
10
10
10
11
11
11
13
11
11
11
10
13
13
13
13
13
13
10
11
10
11
13
11
10
11
1
1
1
1
1
1
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
ATCC_27673
AD011
ATCC_27536
MCC_1489
LAC
MCC_0483
UCC2003 ATCC25527
ATCC_27672
MCC_1604
MCC_1605
MCC_1340
MCC_1454
MCC_1128
ANI
MCC_1114
MCC_1094
MCC_0476
MCC_0305
MCC_0121
CECT7263
DSM20213
ACS_071_V_Sch8
NCC2705
ATCC15697 JDM301
KACC91563
F8
JCM1217
DJO10A
BBMN68
CCUG52486
ATCC55813
35B
44B
157F
1_6B
2_2B
CAZy family
BRE
MCC_0499
INF
LON
11
3
4
4
4
3
3
2
3
4
3
3
3
3
0
1
0
0
0
2
0
2
2
1
1
1
1
1
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
1
1
2
2
1
2
2
2
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
13
11
11
13
6
11
7
12
10
6
7
7
8
1
6
1
0
0
1
0
0
0
0
0
0
1
0
1
0
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
5
6
6
5
4
5
3
0
5
0
0
0
5
1
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
1
1
1
1
0
0
0
0
1
0
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
1
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
0
1
1
1
1
1
0
1
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
Figure 3: Distribution of genes involved in the metabolism of plant-derived sugars predicted by the CAZy database. The abbreviations used for species/subspecies are as follows: INF, B. longum subsp. infantis; LON, B. longum subsp. longum; BRE, B. breve; ANI, B. animalis subsp. animalis; LAC, B. animalis subsp. lactis.
and is further metabolised [34]. In the current study, a sevengene operon involved in this pathway was observed in nearly all strains of B. longum and B. breve and in three strains of B. animalis subsp. animalis. This distribution is in complete accord with results regarding in vitro LNB utilisation [35]. A previous report revealing a strong ability to grow in human milk also supports these claims [36]. 3.3.2. Mucin and N-Glycans. Another substrate for this metabolic pathway is GNB, which is a core structure of gastrointestinal mucin. Mucins are extensively O-glycosylated proteins and are thought to serve as a potential carbon source for gut microbiota. Studies have revealed that the main core structures of gastric/duodenal and intestinal mucins are core1, 2-type and core-3-type O-glycans, respectively [37, 38]. A homologous gene encoding 𝛼-N-acetylgalactosaminidase, acting on core-3-type O-glycan (NagBb) [39], was observed in 2 strains of B. longum subsp. infantis, 11 strains of B. longum subsp. longum, and 13 strains of B. breve in the present study. In contrast, a gene encoding endo-𝛼N-acetylgalactosaminidase, which is predicted to release GNB-containing glycans from core-1-type O-glycan mucin (BLD 1258) [40], was observed only in B. longum subsp. longum. In addition, the terminal ends of glycoconjugates in the suckling gut have been reported to predominantly consist of sialic acid, whereas in the adult, they predominantly consist of fucose [41, 42]. In our study, nearly all strains of B. longum subsp. infantis and B. breve contained a gene encoding sialidase, which might be useful for this species to colonise the infant intestine. Most strains of B. longum and B. breve, but not B. animalis strains, were found to possess genes involved in the pathway for the utilisation of N-acetylglucosamine (GlcNAc) and GalNAc, which are the major constituents of host glycans. Furthermore, gene homologues encoding endo-betaN-acetylglucosaminidase (BLD 0197) [43], which releases complex N-glycans from human milk glycoproteins, and alpha-mannosidase (Blon 0868, 0869) were found in certain strains of these two species. These enzymes involved in the
degradation of host glycans might play a role in the utilisation of intrinsic carbohydrate sources. Bifidobacteria are generally residents of the intestines of animals, including warm-blooded mammals and social insects, and several bifidobacterial species are typical inhabitants of the human gut (designated human-residential bifidobacteria or HRB). All strains of B. longum and B. breve, which are typical HRB, possess certain genes related to the pathway for the utilisation of LNT/LNB, which are core structures of type I oligosaccharides that are specific to human breast milk (Figure 2). Among HRB strains in our study, nearly all possessed gene operons involved in the GNB/LNB pathway, whereas genes upstream of HMO utilisation, such as fucosidase, sialidase, and LNBase, were species/strain dependent. These results suggest that each HRB strain might evolve to assimilate HMOs, for which hundreds of types of structures have been reported [44]. In contrast, B. animalis is a non-HRB species; although certain strains of B. animalis subsp. lactis have also been isolated from humans, they have been found in faecal samples but rarely in colon mucosal samples [27, 45]. In addition, previous reports have shown that B. animalis is a strictly monophyletic group, and the evolutionary distance between B. animalis and species of HRB was shown to be relatively far based on 16S rRNA multigene alignments and comparative genomics [11, 13, 27, 46]. Our results indicate that there are fewer homologues involved in the degradation of host glycan by B. animalis (Figure 2). These results suggest that the observed differences in the gene distribution might be the result of the adaptation of these strains to their residential environments. 3.4. Carbohydrate-Active Enzymes for Plant-Derived Sugars. Regarding extrinsic carbohydrates, a variety of resistant fibres, which can be dietary compounds, are delivered to the colon, where bifidobacteria reside. Based on CAZy classification, all of the strains possessed a large number of homologous genes encoding GH 13 family members (Figure 3), which has previously been reported as a characteristic feature of bifidobacterial genomes [47, 48].
8
International Journal of Genomics
INF
1_6B 2_2B 35B 44B 157F ATCC55813 BBMN68 CCUG52486 DJO10A F8 JCM1217 KACC91563 NCC2705
KO assignment ABC transporters, prokaryotic type K02050 ABC.SN.P; NitT/TauT family transport system permease protein K02049 ABC.SN.A; NitT/TauT family transport system ATP-binding protein K15553 ssuA; sulfonate transport system substrate-binding protein K15554 ssuC; sulfonate transport system permease protein K15555 ssuB; sulfonate transport system ATP-binding protein [EC:3.6.3.-] K02055 ABC.SP.S; putative spermidine/putrescine transport system substrate-binding protein K02053 ABC.SP.P; putative spermidine/putrescine transport system permease protein K02054 ABC.SP.P1; putative spermidine/putrescine transport system permease protein K02052 ABC.SP.A; putative spermidine/putrescine transport system ATP-binding protein K05845 opuC; osmoprotectant transport system substrate-binding protein K05847 opuA; osmoprotectant transport system ATP-binding protein K10108 malE; maltose/maltodextrin transport system substrate-binding protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K15770 cycB, ganO; arabinogalactan oligomer/maltooligosaccharide transport system substrate-binding protein K15771 ganP; arabinogalactan oligomer/maltooligosaccharide transport system permease protein K15772 ganQ; arabinogalactan oligomer/maltooligosaccharide transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K10117 msmE; raffinose/stachyose/melibiose transport system substrate-binding protein K10118 msmF; raffinose/stachyose/melibiose transport system permease protein K10119 msmG; raffinose/stachyose/melibiose transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K10233 aglF, ggtC; alpha-glucoside transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K17317 gtsC, glcG; glucose/mannose transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K10236 thuE; trehalose/maltose transport system substrate-binding protein K10200 ABC.NGC.S; N-acetylglucosamine transport system substrate-binding protein K10201 ABC.NGC.P; N-acetylglucosamine transport system permease protein K10202 ABC.NGC.P1; N-acetylglucosamine transport system permease protein K10242 cebG; cellobiose transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K17331 dasC; N, N-diacetylchitobiose transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K17245 chiF; putative chitobiose transport system permease protein K10188 lacE, araN; lactose/L-arabinose transport system substrate-binding protein K10189 lacF, araP; lactose/L-arabinose transport system permease protein K17318 K17318, lplA; putative aldouronate transport system substrate-binding protein K17319 lplB; putative aldouronate transport system permease protein K17320 lplC; putative aldouronate transport system permease protein K10546 ABC.GGU.S, chvE; putative multiple sugar transport system substrate-binding protein K10547 ABC.GGU.P, gguB; putative multiple sugar transport system permease protein K10548 ABC.GGU.A, gguA; putative multiple sugar transport system ATP-binding protein K10555 lsrB; AI-2 transport system substrate-binding protein K10559 rhaS; rhamnose transport system substrate-binding protein K10439 rbsB; ribose transport system substrate-binding protein K10440 rbsC; ribose transport system permease protein K10441 rbsA; ribose transport system ATP-binding protein [EC:3.6.3.17] K17238 inoF; inositol-phosphate transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K05813 ugpB; sn-glycerol 3-phosphate transport system substrate-binding protein K05814 ugpA; sn-glycerol 3-phosphate transport system permease protein K05815 ugpE; sn-glycerol 3-phosphate transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K17234 araN; arabinosaccharide transport system substrate-binding protein K17236 araQ; arabinosaccharide transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K02027 ABC.MS.S; multiple sugar transport system substrate-binding protein K02025 ABC.MS.P; multiple sugar transport system permease protein K02026 ABC.MS.P1; multiple sugar transport system permease protein K10112 msmX, msmK, malK, sugC, ggtA, msiK; multiple sugar transport system ATP-binding protein K02058 ABC.SS.S; simple sugar transport system substrate-binding protein K02057 ABC.SS.P; simple sugar transport system permease protein K02056 ABC.SS.A; simple sugar transport system ATP-binding protein [EC:3.6.3.17] K02069 ABC.X2.P; putative ABC transport system permease protein K02068 ABC.X2.A; putative ABC transport system ATP-binding protein K02040 pstS; phosphate transport system substrate-binding protein K02037 pstC; phosphate transport system permease protein K02038 pstA; phosphate transport system permease protein K02036 pstB; phosphate transport system ATP-binding protein [EC:3.6.3.27] K02044 phnD; phosphonate transport system substrate-binding protein K02042 phnE; phosphonate transport system permease protein K02041 phnC; phosphonate transport system ATP-binding protein [EC:3.6.3.28] K10005 gluB; glutamate transport system substrate-binding protein K10006 gluC; glutamate transport system permease protein K10007 gluD; glutamate transport system permease protein K10008 gluA; glutamate transport system ATP-binding protein [EC:3.6.3.-] K02424 fliY; cystine transport system substrate-binding protein K10009 ABC.CYST.P; cystine transport system permease protein K10010 ABC.CYST.A; cystine transport system ATP-binding protein [EC:3.6.3.-] K16958 tcyL; L-cystine transport system permease protein K01999 livK; branched-chain amino-acid transport system substrate-binding protein K01997 livH; branched-chain amino-acid transport system permease protein K01998 livM; branched-chain amino-acid transport system permease protein K01995 livG; branched-chain amino-acid transport system ATP-binding protein K01996 livF; branched-chain amino-acid transport system ATP-binding protein K11959 urtA; urea transport system substrate-binding protein K11960 urtB; urea transport system permease protein K11961 urtC; urea transport system permease protein K11962 urtD; urea transport system ATP-binding protein K11963 urtE; urea transport system ATP-binding protein K02073 metQ; D-methionine transport system substrate-binding protein K02072 metI; D-methionine transport system permease protein K02071 metN; D-methionine transport system ATP-binding protein K16961 yxeM; putative amino-acid transport system substrate-binding protein K16962 yxeN; putative amino-acid transport system permease protein K02030 ABC.PA.S; polar amino-acid transport system substrate-binding protein K02029 ABC.PA.P; polar amino-acid transport system permease protein K02028 ABC.PA.A; polar amino-acid transport system ATP-binding protein [EC:3.6.3.21] K15580 oppA, mppA; oligopeptide transport system substrate-binding protein K15581 oppB; oligopeptide transport system permease protein K15582 oppC; oligopeptide transport system permease protein K10823 oppF; oligopeptide transport system ATP-binding protein K02035 ABC.PE.S; peptide/nickel transport system substrate-binding protein K02033 ABC.PE.P; peptide/nickel transport system permease protein K02034 ABC.PE.P1; peptide/nickel transport system permease protein K02031 ABC.PE.A; peptide/nickel transport system ATP-binding protein K02032 ABC.PE.A1; peptide/nickel transport system ATP-binding protein K02013 ABC.FEV.A; iron complex transport system ATP-binding protein [EC:3.6.3.34] K02006 cbiO; cobalt/nickel transport system ATP-binding protein K02006 cbiO; cobalt/nickel transport system ATP-binding protein K03523 bioY; biotin transport system substrate-specific component K16783 bioN; biotin transport system permease protein K16784 bioM; biotin transport system ATP-binding protein [EC:3.6.3.-] K03523 bioY; biotin transport system substrate-specific component K16923 qrtT; energy-coupling factor transport system substrate-specific component K16925 ykoE; energy-coupling factor transport system substrate-specific component K16926 htsT; energy-coupling factor transport system substrate-specific component K16785 ecfT; energy-coupling factor transport system permease protein K16786 ecfA1; energy-coupling factor transport system ATP-binding protein [EC:3.6.3.-] K16787 ecfA2; energy-coupling factor transport system ATP-binding protein [EC:3.6.3.-] K02077 ABC.ZM.S; zinc/manganese transport system substrate-binding protein K02075 ABC.ZM.P; zinc/manganese transport system permease protein K02074 ABC.ZM.A; zinc/manganese transport system ATP-binding protein K09810 ABC.LPT.A, lolD; lipoprotein-releasing system ATP-binding protein [EC:3.6.3.-] K09811 ftsX; cell division transport system permease protein K09812 ftsE; cell division transport system ATP-binding protein K02004 ABC.CD.P; putative ABC transport system permease protein K02003 ABC.CD.A; putative ABC transport system ATP-binding protein K01992 ABC-2.P; ABC-2 type transport system permease protein K01990 ABC-2.A; ABC-2 type transport system ATP-binding protein K09013 sufC; Fe-S cluster assembly ATP-binding protein
BRE
ANI
LAC
ATCC15697 JDM301 ACS_071_V_Sch8b CECT7263 DSM20213 MCC_0121 MCC_0305 MCC_0476 MCC_1094 MCC_1114 MCC_1128 MCC_1340 MCC_1454 MCC_1604 MCC_1605 UCC2003 ATCC25527 ATCC_27672 MCC_0483 MCC_0499 MCC_1489 AD011 ATCC_27536 ATCC_27673 ATCC_27674 B420 BB_12 Bi_07 Bl12 Bl_04 BLC1 BS01 CNCMI_2494 DSM10140 HN019 V9
LON
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Figure 4: Continued.
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International Journal of Genomics
9
Major facilitator superfamily (MFS) K02100 araE; MFS transporter, SP family, arabinose: H+ symporter K08138 xylE; MFS transporter, SP family, xylose: H+ symporter K08139 HXT; MFS transporter, SP family, sugar: H+ symporter K06610 iolF; MFS transporter, SP family, inositol transporter K02532 lacY; MFS transporter, OHS family, lactose permease K02429 fucP; MFS transporter, FHS family, L-fucose permease K06901 pbuG; putative MFS transporter, AGZA family, xanthine/uracil permease K03762 proP; MFS transporter, MHS family, proline/betaine transporter K08177 oxlT; MFS transporter, OFA family, oxalate/formate antiporter K08151 tetA; MFS transporter, DHA1 family, tetracycline resistance protein K08156 araJ; MFS transporter, DHA1 family, arabinose polymer transporter K08166 mmr; MFS transporter, DHA2 family, methylenomycin A resistance protein K08167 smvA; MFS transporter, DHA2 family, methyl viologen resistance protein SmvA K08168 tetB; MFS transporter, DHA2 family, metal-tetracycline-proton antiporter K08217 mef; MFS transporter, DHA3 family, macrolide efflux protein K06902 UMF1; MFS transporter, UMF1 family K08369 ydjE; MFS transporter, putative metabolite: H+ symporter Phosphotransferase system (PTS) K08483 PTS-EI.PTSI, ptsI; phosphotransferase system, enzyme I, PtsI [EC:2.7.3.9] K11189 PTS-HPR; phosphocarrier protein K02777 PTS-Glc-EIIA, crr; PTS system, glucose-specific IIA component [EC:2.7.1.69] K02803 PTS-Nag-EIIB, nagE; PTS system, N-acetylglucosamine-specific IIB component [EC:2.7.1.69] K02804 PTS-Nag-EIIC, nagE; PTS system, N-acetylglucosamine-specific IIC component K02777 PTS-Glc-EIIA, crr; PTS system, glucose-specific IIA component [EC:2.7.1.69] K02756 PTS-Bgl-EIIB, bglF; PTS system, beta-glucosides-specific IIB component [EC:2.7.1.69] K02757 PTS-Bgl-EIIC, bglF; PTS system, beta-glucosides-specific IIC component K02777 PTS-Glc-EIIA, crr; PTS system, glucose-specific IIA component [EC:2.7.1.69] K02777 PTS-Glc-EIIA, crr; PTS system, glucose-specific IIA component [EC:2.7.1.69] K02777 PTS-Glc-EIIA, crr; PTS system, glucose-specific IIA component [EC:2.7.1.69] K02777 PTS-Glc-EIIA, crr; PTS system, glucose-specific IIA component [EC:2.7.1.69] K02768 PTS-Fru-EIIA, fruB; PTS system, fructose-specific IIA component [EC:2.7.1.69] K02769 PTS-Fru-EIIB, fruA; PTS system, fructose-specific IIB component [EC:2.7.1.69] K02770 PTS-Fru-EIIC, fruA; PTS system, fructose-specific IIC component K11202 PTS-Fru2-EIIB; PTS system, fructose-specific IIB-like component [EC:2.7.1.69] K02773 PTS-Gat-EIIA, gatA; PTS system, galactitol-specific IIA component [EC:2.7.1.69] K02775 PTS-Gat-EIIC, gatC; PTS system, galactitol-specific IIC component K02821 PTS-Ula-EIIA, ulaC, sgaA; PTS system, ascorbate-specific IIA component [EC:2.7.1.69] K02822 PTS-Ula-EIIB, ulaB, sgaB; PTS system, ascorbate-specific IIB component [EC:2.7.1.69] K03475 PTS-Ula-EIIC, ulaA, sgaT; PTS system, ascorbate-specific IIC component K02806 PTS-Ntr-EIIA, ptsN; PTS system, nitrogen regulatory IIA component [EC:2.7.1.69] Other transporters K06188 aqpZ; aquaporin Z K02440 GLPF; glycerol uptake facilitator protein
ANI
LAC
AD011 ATCC_27536 ATCC_27673 ATCC_27674 B420 BB_12 Bi_07 Bl12 Bl_04 BLC1 BS01 CNCMI_2494 DSM10140 HN019 V9
BRE
ACS_071_V_Sch8b CECT7263 DSM20213 MCC_0121 MCC_0305 MCC_0476 MCC_1094 MCC_1114 MCC_1128 MCC_1340 MCC_1454 MCC_1604 MCC_1605 UCC2003 ATCC25527 ATCC_27672 MCC_0483 MCC_0499 MCC_1489
KO assignment
INF
1_6B 2_2B 35B 44B 157F ATCC55813 BBMN68 CCUG52486 DJO10A F8 JCM1217 KACC91563 NCC2705 ATCC15697 JDM301
LON
0
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Figure 4: Distribution of genes encoding membrane transporters for carbohydrate. The abbreviations used for species/subspecies are as follows: INF, B. longum subsp. infantis; LON, B. longum subsp. longum; BRE, B. breve; ANI, B. animalis subsp. animalis; LAC, B. animalis subsp. lactis.
These enzymes are typical enzymes for the degradation of alpha-glucopyranose units such as pullulan, starch, and amylopectin. In particular, B. longum subsp. longum was shown to be genetically well equipped for the fermentation of plant-derived sugars (Figure 3), which are assumed to be not introduced into the infant gut before weaning. A large number of GH 43 and 51 family members, which are enzymes responsible for the degradation of arabinose/xylose units such as arabinofuranoside and xylan, were found to be specific to B. longum subsp. longum, with strain specificity for several of these genes. Taken together with the distribution of genes for host glycan utilisation, such a wide range of genes for carbohydrate utilisation provides an advantage for colonisation of the human intestine by B. longum subsp. longum. This result might explain why only B. longum subsp. longum is a predominant species in both the infant and the adult intestines [49]. 3.5. Membrane Transporters for Carbohydrate. KO assignment indicated several differences in the distribution of carbohydrate transporters at the subspecies level. The subspecies that possessed the greatest number of transporter homologues was B. longum subsp. infantis (83.5 ± 2.1), including taxon-specific transporters for GlcNAc, phosphonate, and urea, whereas B. animalis subsp. lactis possessed the lowest number of these homologues (33.7±1.3). All three species can assimilate fructose; however, only species of B. longum exhibited a set of high-affinity fructose-specific ABC transporters (K02056, K02057, and K02058), which have been reported to be involved in efficiently converting fructose to acetate under certain gut conditions [5]. B. longum subsp. longum
lacked a homologous gene set for arabinogalactan transport. However, twelve strains of B. longum subsp. longum possessed a set of homologous genes encoding extracellular enzymes for the degradation of arabinogalactan (BLLJ 1840) [50]. B. breve also exhibited certain unique ABC transporters for lactose/Larabinose, ribose, and sn-glycerol 3-phosphate. We found distinctive differences between the three species in terms of the distribution of genes related to the phosphotransferase system (PTS), which is known to be a multicomponent system specific for sugar uptake that operates in global carbon regulation in many bacteria [51]. Strains of B. longum and B. breve possessed three homologues, encoding phosphoenolpyruvate-protein kinase, phosphocarrier protein, and beta-glucoside-PTS, whereas none of the B. animalis strains exhibited these genes. Additionally, nearly all strains of B. breve possessed homologues of Nacetylglucosamine, glucose, fructose, and ascorbate, which are involved in the PTS (Figure 4).
4. Conclusions Certain strains belonging to the three bifidobacterial species targeted in this study have been reported to exert numerous beneficial effects on human health as probiotics. Probiotic functionality is generally thought to be strain dependent. In fact, our analysis confirmed that certain genes, such as those encoding membrane transporters and enzymes for host glycan utilisation, are strain dependent. However, our data also demonstrated that there are common characteristics in each species that may be important in light of the species’ health-promoting effects on their hosts. Additionally,
10 differences in characteristics might be a result of adaptation to the nutrient environments of each species (such as HRB versus non-HRB). Our results support previous observations based on the investigation of certain type strains of bifidobacterial species and enable the qualification of several of these characteristics as species common or strain specific [33, 39, 50, 52]. Taken together with other characteristics, such as vitamin metabolism [4, 53], colonisation factors [54], and extracellular components [55, 56], we believe that these findings will help to predict the features of probiotic strains. However, further study is needed to evaluate other non-HRB and HRB bifidobacterial species to attain a better understanding of the characteristics of these bacteria as well as the mechanisms underlying their residence in the host intestine and their potential functions as probiotics.
Conflict of Interests All authors are employees of Morinaga Milk Industry Co., Ltd., which has several probiotic products marketed worldwide.
Acknowledgments The authors wish to thank Kensuke Suzuki and Keiichiro Nishihama (Illumina Inc.) for technical support.
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